Difference between revisions of "Lidstrom:Preparing Freezer Cell Stocks"

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==DMSO==
 
==DMSO==
 
*Grow ~ 5 mL of E. Coli overnight with appropriate antibiotics.
 
*Grow ~ 5 mL of E. Coli overnight with appropriate antibiotics.
*Centrifuge for 5-10 minutes & pour off supernatant.
+
*Centrifuge for ~2+ minutes & pour off supernatant.
*Resuspend in 900 uL of HY (hypho) and 100 uL of filter-sterilized DMSO.  Hypho is better than LB for this, I hear it buffers.
+
*Resuspend in 900 uL of HY (hypho) and 100 uL of filter-sterilized DMSO.  Hypho is better than LB for this; I hear it buffers.
 +
** ''' must use a filter that won't dissolve in DMSO '''.  Most of the filters we have are not appropriate.  
 
*Store at -80oC.
 
*Store at -80oC.
 
*Test stock after frozen a while.
 
*Test stock after frozen a while.
 +
 +
=== Filtering DMSO: don't dissolve the filter ===
 +
* Pure DMSO dissolves many of the filters common in our lab.
 +
* "To prepare a sterile solution, use a teflon or nylon membrane to sterile-filter the DMSO; do not use a cellulose acetate membrane." [http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/d8779pis.Par.0001.File.tmp/d8779pis.pdf Sigma/Source]
  
 
==Glycerol==
 
==Glycerol==
 
*
 
*

Latest revision as of 20:15, 6 June 2013

Back to protocols

DMSO

  • Grow ~ 5 mL of E. Coli overnight with appropriate antibiotics.
  • Centrifuge for ~2+ minutes & pour off supernatant.
  • Resuspend in 900 uL of HY (hypho) and 100 uL of filter-sterilized DMSO. Hypho is better than LB for this; I hear it buffers.
    • must use a filter that won't dissolve in DMSO . Most of the filters we have are not appropriate.
  • Store at -80oC.
  • Test stock after frozen a while.

Filtering DMSO: don't dissolve the filter

  • Pure DMSO dissolves many of the filters common in our lab.
  • "To prepare a sterile solution, use a teflon or nylon membrane to sterile-filter the DMSO; do not use a cellulose acetate membrane." Sigma/Source

Glycerol